Substrate for cell structures

ABSTRACT

In an apparatus for culturing cells which comprises a plate-like body with a lattice structure having openings separated from one another by sidewalls and a bottom disposed on one side of the lattice structure and having passages permeable for liquids but not for cells so as to form, with the lattice structure, cavities for receiving cells to be grown therein, the cavities have a clear width of between 50 μm and 1000 μm and the bottom consists of a material to which cells placed into the cavities will not easily attach so that the cells grow from the side walls of the cavities causing them to form a three-dimensional cell structure.

The present application is a continuation-in-part application ofinternational application PCT/DE92/00815 filed Sep. 23, 1992 andclaiming the priority of German Patent Application P4132379.3 of Sep.28, 1991.

BACKGROUND OF THE INVENTION

The invention relates to an apparatus for culturing cells comprising alattice-like structure with cavities separated by walls and a bottomclosing the cavities between the walls and being permeable for liquidsbut not for cells.

Such a substrate is known from DE 29 02 026 A1. The substrate disclosedtherein comprises a lattice structure with a bottom which is removablyconnected to the lattice structure. The lattice structure consists of aplate of plastic material which includes a plurality of passages(chambers) separated from one another by webs. The chambers have a clearwidth of about 10 mm (10,000 μm).

The bottom may consist of a glass plate or preferably a plastic plate orplastic foil. With this substrate the cells develop on the bottomthereby generating essentially two-dimensional cell cultures. It isfurther pointed out that the bottom with the cell cultures thereon formsa microscopic object carrier or that it may be disposed on an objectcarrier of glass. This again assumes that the cells grow essentiallyonly on the bottom and there, form an essentially two-dimensional layer.

From the publication "Morphological examinations with Fallopian tubeepithelial tissue cells of the rabbit: Primary cultures of polycarbonatemembranes" by I. G. Noske in TECNOMARA NEWS IV/90, LabortechnischeInformationsschrift, a two-dimensional substrate for cell cultures and atwo-dimensional cell culture are known. The substrate consists of amembrane with pores of different sizes. Some of these pores extendthrough the membrane. The cells have a tendency to anchor themselves inthese pores. Essentially, the cells form a mono layer which covers theupper and lower surfaces of the membrane.

Such substrates have the advantage that a nutrient solution can easilybe supplied to the cells and metabolic waste can easily be removed.Also, cells attached to such substrates can be observed optically underthe microscope. It is however pointed out in this publication that thecells vary in shape depending on the kind of substrate utilized. Theygrow, for example, extremely flat on a glass or plastic bottom so thatthe polarity which is so important for their functions is notguaranteed.

In the article "Solid tissue masses formed in vitro from cells culturedon artificial capillaries" by Richard A. Knazek, Federation Proceedings,vol. 33, No. 8 (August 1974) and in a company brochure of the company"dunn Labortechnik GmbH" in Asbach, Germany, under the title "Cellmax™100-Hollow Fiber Bioreactor System for Mammalian and Insect CellCulture" which refers to the Knazek publication, disclose a cellsubstrate which consists of a plurality of parallel capillaries arrangedspaced from one another. The capillaries are combined at one end by aconnecting member via which nutrient solution can be conducted throughthe capillaries. The cells grow on the outside of the capillaries in thespaces formed therebetween. The nutrient solution diffuses from insidethe capillaries through the walls of the capillaries into the spacestherebetween for feeding the cells. In this manner, a three-dimensionalcell structure is generated.

However, for different reasons, such a substrate is not very suitablefor research with cell cultures. The metabolic gradient is not clearlydefined because of the irregular geometry. Furthermore, the diffusion ofcompounds is always limited to certain molecular weights depending onthe material used for the various capillaries. For some applications acoating of the substrate is necessary; such a coating may act as adiffusion barrier with this kind of cell substrate which would result ina collapse of the metabolic gradient. Finally, such a substrate cannotbe arranged in such a manner that microscopic observation of the cellsduring cultivation is possible.

A further substrate for the cultivation of human and/or animal cells isdisclosed in EP 0 205 790 B1. This substrate comprises a compositestructure of pearl-like particles with a diameter of 50 to 300 μm withinterstices in which three-dimensional cell cultures can develop. Thepearl-like particles have on their surfaces macropores with diameters ofless than 10 μm, preferably in the range of 0.1 to 3 μm. The macroporesform a capillary system, into which the cells cannot grow since thediameter of the cells used herein is about 20 μm. However nutrients canbe supplied through this system of capillaries and metabolic waste canbe removed.

Nevertheless, such a substrate has essentially the same disadvantage asthe capillary tube substrate.

FR-A2522014 discloses a plate-like substrate obtained by a moldingprocess which has a plurality of cavities of a width of 10 to 1500 μm;it includes a bottom which is impermeable for liquids.

It is the object of the present invention to provide a cell substratewithout the disadvantages mentioned above. Particularly, the advantagesof membrane-like substrates are to be retained while the growth ofthree-dimensional cell structures is promoted.

SUMMARY OF THE INVENTION

In an apparatus for culturing cells which comprises a plate-like bodywith a lattice structure having openings separated from one another bysidewalls and a bottom disposed on one side of the lattice structure andhaving passages permeable for liquids but not for cells so as to form,with the lattice structure, cavities for receiving cells to be growntherein, the cavities have a clear width of between 50 μm and 1000 μmand the bottom consists of a material to which cells placed into thecavities will not easily attach so that the cells grow from the sidewalls of the cavities causing them to form a three-dimensional cellstructure.

The openings in the lattice structure, in principle, may have any shape.Preferably, they are of rectangular or hexagonal shape because suchstructures can be formed with webs which have all the same length. Withother, also for example, irregular shapes, the clear width should be inthe given range in every direction. However, the clear width does notneed to be constant. The openings may have, for example, the shape ofoblong or hexagonal truncated pyramids such that they become narrowertoward the bottom wherein the clear width as given should be maintainedin any cross-sectional plane parallel to the bottom.

In spite of the fact that the substrate according to the invention hasessentially the same shape as the one disclosed in DE 29 02 026 referredto above, the dimensions of the substrate according to the inventionprovide for essential differences in its effectiveness and in the waythe cell cultures can grow on it. The dimensions of the substrateaccording to the invention are so selected that the cells disposedthereon do not (like in the known substrate described above) grow ontothe bottom but anchor themselves to the side walls and produce a largenumber of interconnections in horizontal and particularly in verticaldirections. In this manner, a compact three-dimensional cell structureis built up whose size is predetermined by the dimensions of theparticular openings. As a result of the dimensions provided inaccordance with the invention nutrition supply to the cell structures isalso insured.

The basic idea in selecting the dimensions in accordance with theinvention is to provide as little flat bottom surface as possible forthe cells in developing the culture. It has been found that athree-dimensional build-up of the cell structure can no longer beobtained if several cells can attach themselves to the bottom surfaceand form a two-dimensional layer as in the substrate described above.

A substrate with cavities of a clear width in the range of 150 μm to 400μm is preferred. Cavities with these dimensions are particularlysuitable for cells with a diameter of 20 μm.

The depth of the cavities depends on the intended use of the substrateaccording to the invention. It is limited by the physiologicalrequirement for a good nutrition supply for the central part of the cellstructure in any Particular cavity. With this consideration in view, itappears that a depth of between 50 and 300 μm is most appropriate.However, for special nonphysiological applications, a depth in the rangeof 300 to 1000 μm appears to be more suitable. Such applications are,for example, experiments with tumor models wherein a central necroticzone is intentionally generated.

Particularly preferred are substrates whose cavities have the shape of atruncated pyramid becoming smaller toward their bottom. Such substratescan be particularly easily produced by mechanical micromanufacturingprocedures.

With a suitable selection of the width of the webs on the open latticeside, which is not covered by the bottom of the plate-like latticestructure, a well-defined cell structure layer can be produced on a verysmall area. In this arrangement the cells grow out of the cavities andjoin to form a cell structure which covers the whole substrate and whichdoes not contain any objectionable support structure. To achieve this,it has been found that the width of the webs should be in the range of15 to 115 μm.

With the substrate according to the invention the bottom is requiredactually only for the planting of the cell culture. Since, as a resultof the dimensions provided in accordance with the invention, the cellsanchor themselves to the walls of the cavities, it can be advantageous,particularly for microscopic examination of the cell culture, if thebottom is removable. This is possible since, in contrast to the priorart substrate referred to above, the cell culture is not connected tothe bottom but to the walls of the cavities formed in the latticestructured plate-like substrate.

The bottom may comprise a microporous plate which is permeable only forliquids but not for any cells such as a ceramic frit provided with arespective plastic membrane. However, a lattice-like bottom plate withwell-defined openings separated by walls is preferred. The sizes of theopenings can be such that even single cells are prevented from passingtherethrough.

In any case, the bottom is, in accordance with the invention, sodesigned that the cells placed into the cavities, essentially, cannotattach themselves to the bottom.

This can be achieved by a structure wherein the bottom does not have aflat area sufficiently large to accommodate a cell. If, for example, aceramic frit is used as a bottom, the porosity of the frit should beselected accordingly. However, the bottom may also be a plate or foilwhich consists of a material that is not suitable for the attachment ofcells.

The plates or foils used as bottoms for the cell substrates in the knownarrangements have free surface areas between the pores whose widths aregenerally within a certain range. A more even-sized bottom can be madewith a regular lattice structure with openings separated by walls. Sucha lattice structure can also be made by the methods ofmicromanufacturing (mechanical micromanufacture, X-ray depth lithographyor other lithographic procedures).

Particularly preferred are lattice-like bottom plates which, inprinciple, do not provide sufficient flat area for the cells placedthereon for attachment. To this end, the walls of the lattice structureutilized which are maximally 200 μm thick are in the range of 1 to 20 μmthick and the clear width of the openings, which is maximally 5 μm, isgenerally in the range of 1 to 5 μm. The lower limit of the dimensionsof such a lattice structure will generally be limited by themanufacturing method. In the interest of unimpeded supply of nutrientsolution and for appropriate removal of metabolic waste, the openingarea in the bottom is larger than the bottom surface area occupied bythe webs of the lattice structures.

It is advantageous if the substrate according to the invention consistsof a transparent material. As a transparent materialpolymethylmethacrylate (PNMA) is particularly suitable. Substrates ofPNNA can be easily made by micromanufacturing methods and PPMA hasproperties suitable for cell culture growth thereon.

Microscopic examinations are facilitated if the bottom plate can beremoved from the lattice structure after establishment of the cellculture. If the substrate consists of a lattice structure plate withremovable bottom it is sufficient if the lattice structure is made of atransparent material.

In some applications, the inner wall is of the cavities and the exposedsurface areas of the bottom are coated in such a manner that the cellsdo not attach to the surfaces. In that case the substrate according tothe invention is suitable not only for growing predetermined cellculture layers but also for the build-up of multicellular spheroids of apredetermined very uniform size. A coating suitable for this purpose is,for example, a silicon coating. With such a substrate, the cellsdeposited thereon aggregate in the various cavities. Then the clearwidth of the cavities determines the diameter of the spheroids growntherein.

The substrate according to the invention is, for example, a plate-likebody which includes lattice-like structured square cavities disposedclosely adjacent one another with a cross-section which becomes smallerwith depth in the shape of an inversed truncated pyramid. At the bottomof the cavities there are substantially smaller openings in the shape ofa truncated pyramid which openings extend through the bottom of theplate-like body. In this embodiment the plate-like body has an integralbottom which cannot be removed.

In another embodiment the substrate according to the invention comprisesan open-ended lattice structure disposed on a microporous bottom plate,particularly a micro lattice bottom plate.

The three-dimensional cultivation of cells in the substrate according tothe invention provides for cell differentiation in addition to thesubstrate contacts via cell-to-cell interactions. In addition tomiroscopic observations series-slices of the substrate with the cellsdisposed therein can be prepared which is a distinctive advantage forthe application of histological and histochemical methods.

The substrate according to the invention may be integrated into anobject carrier or a medium circulation system. With multilayer cellgrowth tight to the walls of the lattice structure, the substraterepresents a kind of fabric filter with two physiological semispacesdefined at opposite sides thereof. With different medium admission inthe circulation system, vertically oriented metabolic gradients developin the culture which are not blocked or inhibited by additional layerformation on the lattice structure (for example, with components of theextra cellular matrix with cell-specific applications).

The substrate according to the invention represents a rigid supportstructure for cells and facilitates the preparation of well-definedlayers of various types of cells. As a result of the heterotypical cellinteraction occurring in this connection processes cafferentiationprocesses can occur. It is, for example, possible to study cell-specifictransport processes with such systems. Specifically, the functions ofthe organ system "skin" whose restitution in vitro is difficult, can bestudied more closely by passing air through one of the semispaces as amedium.

Such a closely organ-like culture technique on the basis of thesubstrate according to the invention can be used with a large number ofdifferent types of cells. It can be utilized for the studying ofphenomena in many biomedical areas wherein a high measure of celldifferentiation is required:

toxicological examinations under near real conditions;

in the development of medications:

effectiveness and restitution testing;

in basic research;

as an alternative to animal experiments, particularly in connection withexaminations of complicated organ systems (for example, the liver or theskin).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an embodiment of the substrate with square openings andlattice webs 2 with a porous bottom plate 3;

FIG. 2a shows schematically the arrangement of FIG. 1;

FIG. 2b shows schematically an arrangement with webs of invertedV-shaped cross-section;

FIGS. 3 to 5 show different steps for a mold insert with which anembodiment of the substrate according to the invention can be formed,specifically

FIG. 3a shows the prepared mold insert;

FIG. 3b is a side view of the mold of FIG. 3a;

FIG. 4 shows the roughly structured mold insert;

FIG. 5 shows the finely structured mold insert;

FIG. 6 shows a lattice obtained with the roughly structured mold;

FIG. 7 shows a web structure molded with the finely structured moldinsert with small depressions at the bottom of the cavities separated bythe webs from which then the passages are formed;

FIG. 8 shows the underside of the substrate from which a layer was cutoff to form passages from the depressions;

FIG. 9 is a top view of the substrate of FIG. 8;

FIGS.10a-10e show a microscope objective carrier into which a substrateis integrated; and

FIGS. 11a-11e show another microscope objective carrier with integratedsubstrate.

DESCRIPTION OF THE PREFERRED EMBODIMENTS--EXAMPLES

1. Making a substrate according to the invention

The substrate according to the invention can be made by X-raylithographic procedures or by molding procedures. Below a moldingprocedure is described on the basis of FIGS. 3 to 8 by which a pluralityof the substrates according to the invention can be manufactured inseries in a simple manner.

For the molding of a substrate of a resin material, a mold insert isrequired which is provided with positive structures for forming thecavities and openings of the microstructured substrate.

FIG. 3 shows a premachined mold insert with four raised areas 1 to 4 of10×10 mm in size. The height of the raised areas is 1 mm. Wedge-shapedprofiled diamonds are used to cut intersecting grooves into the topsurface of the raised area in such a manner that a square raster of 400μm square net structure of 280 μm deep grooves of triangularcross-section are formed such that frustopyramidal portions with topsurface areas of 300 μm×300 μm remain between the grooves. This roughstructured shape of the mold insert is shown in FIG. 4.

Subsequently, a substructure of intersecting small grooves is cut intothe top surfaces of the frustopyramidal portion utilizing finerdiamonds: The top surface of each frustopyramidal portion is providedwith a square raster pattern of 40 μm with 8×8 microfrustopyramidalportions. The height of these microfrustopyramidal portions is about 80μm; their top surfaces are about 10 μm². The result of this preparationstep is shown in FIG. 5.

From the mold insert prepared in this manner a plate is formed in aninjection molding machine. FIGS. 6 and 7 show the rough and the finestructure of the molded PNMA plate. In a final manufacturing step thematerial at the bottom of the molded substrate plate is removed; in thismanner, the openings in the bottom of the substrate are exposed. Forthis step, the substrate is fixed on a vacuum mount and material isremoved from the bottom side of the substrate until the openings or thepores are freed. The thickness of the bottom part can be controlled bythe amount of material removed. Since the openings are conical theirsize, that is, their minimum diameter, can also be determined in thismanner. The result of this process step is shown in FIG. 8 which showsthe bottom side of the substrate and in FIG. 9 which shows the top sideof the substrate. If the bottom of the substrate is totally removed, aresin lattice structure of 100 μm wide web portions and mesh aperturesof 300 μm will be obtained which lattice structure may also serve as theplate-like lattice structured substrate.

2. Making a microscope object carrier

A substrate 2 made in the manner described under 1) and having a usablesurface of 10 mm×10 mm is, as shown in FIG. 10, combined with two objectcarrier plates 1 of glass, an adjustment disc 3 and intermediate discs4, which contain temperature control and nutrition channels 5 and 6 toform an object carrier.

The outermost channels 5 serve for the control of the temperature;nutrients are supplied and removed via the intermediate channels 6.

3. Manufacturing of another microscope object carrier

The microscope object carrier consists of two almost identical plates10, 11 (FIGS. 11a-11d) between which a substrate 2 as manufacturedunder 1) is clamped so as to be liquid-sealed therebetween. The upperplate 10 and the lower plate 11 are identical except that four bores areprovided near the edges of the upper plate and the lower plate has fourthreaded openings which are in alignment with the bores and into whichscrews extending through the bores are screwed for clamping thesubstrate between the plates. The Plates consist of brass and arefinished by a galvanically deposited layer of rhodium for contact withthe cell cultures. The outer surface is provided with an oblong recessof a width adapted to receive a commercially available microscope objectcarrier glass plate 14 which is retained by means of two plastic screwsdisposed at each short end of the plate 14 and received in passages 15.The plates are provided with a central opening 16 through which thecells in the substrate can be observed.

Inside, the plates are provided with a circular recess 20 adapted insize to receive the substrate 2.

Two tubes 17 with an inner diameter of 3 mm and an outer diameter of 4mm extend through each plate for connection to a warm water source formaintaining a temperature suitable for cell activity. Center tubes 18,19 extend from opposite sides into the center opening 16. They are usedfor conducting fresh nutrient to the space which is defined by thesubstrate 2 in the middle and outside by the respective glass plates 14,14'. This arrangement when assembled provides for two spaces throughwhich different nutrients can be conducted.

Accordingly, the system as a whole is built-up symmetrically from top tobottom by the following components:

Glass plate 14 screwed onto the upper plate 10 which is screwed to thelower plate 11 with the substrate 2 disposed therebetween to whichanother glass plate 14' is screwed.

With a substrate prepared in accordance with 1) the following biologicalexperiments have been performed in order to demonstrate the growth ofthree-dimensional cell cultures.

4. Placing cells into the substrate

The experiments were performed with two permanent cell lines of themouse: L- and SV40-3T3 cells. Throughout standard growth media wereutilized.

First, the substrates were sterilized by C_(o) -gamma irradiation withabout 100 Gy and were placed into a dry Petri dish. Then 300 but notmore than 400 μl of a concentrated suspension of cells or spheroids(cell aggregates) were placed onto a 1 cm² large lattice structure ofthe substrate. Capillary action caused rapid penetration into thecavities of the substrate within about one minute carrying along thecells or spheroids. After about 15 minutes in an incubator at 37° C.,the Petri dish was carefully filled with medium wherein two variationswere tried out: In one experiment the medium was placed underneath thesubstrate such that the substrate was floating on the medium. In theother, the medium was deposited on top of the substrate. In both casesthe cells in the substrate were fully contacted by the medium. Bothmethods proved to be equal with regard to further growth of the cells inthe incubator. Supplying the nutrient medium to the substrate turned outto be without problems. Providing centrifugal forces for improvingentrance of the cells or spheroids into the substrate was foundunnecessary.

5. Cell affinity of the resin PMMA

A further objective of the experiment was the testing of the affinity ofthe cells regarding the substrate material. The formation ofmicroscopically visible contacts with the substrate walls was taken ascriterium. It appeared that the cells attached themselves to the wallsof the cavities within a few hours. Together with vitality tests andfurther details of cell interaction with the cavity walls, the testdemonstrates that the substrate material PMNA and the substrate geometryprovide for good cell compatibility. The observed high affinity of thecells to the substrate material is possibly partially a result of theirradiation of the substrate for sterilization. The material surfacealterations generated thereby (change of the charge pattern) may favorformation of cell contacts.

6. Vitality and growth behavior of the cells

The objective of the experiment, on one hand, was to identify anycell-damaging effects of the long-time cell growth in the substrate. Onthe other hand, the pattern of interaction of the cells with the cavitywalls of the substrate and their morphologic differentiate on was to betested. In order to identify any cell-damaging effects, cells andspheroids were cultivated in the substrate for more than 2 weeks.Nutrient supply was provided by an exchange of the medium every 2 to 3days. During this period the culture remained sterile; the cellsremained alive and capable of dividing. At the termination of theexperiment all cells were still vital (as proved by a trypanobluediscrimination test) and capable of dividing (proved by a cloningcapability test). Accordingly the system of culturing cells has nocell-damaging effects even over longer periods of time.

For producing and growing several layers of cells in the cavities of thesubstrate, single cells or cell spheroids can be utilized. In practicethe selection is made depending on the properties of the types of cellsused, for example, depending on their proliferation capability.

When single cells are placed into the cavities of a substrate theyattach first to the walls of the cavities and generally in a selectivemanner about in the middle of a wall, that is, at half the height of thewall. Colonization of the porous bottom of a cavity is relatively rare.Starting at the walls the cells multiply without mechanical supporttoward the center of the cavity. A multilayer dense cell structure isformed. This behavior depends to a certain degree on the type of cellused and was particularly apparent with the use of L-fibroblasts.

For testing the behavior of spheroids, SV40-3T3 spheroids with anaverage diameter of 200 μm were placed into the cavities of a substrate.They were smaller than the cavities so that their growth behavior couldbe observed. They first attach themselves to a wall of the cavity. Cellgrowth then increases the volume of the spheroids so that, after a fewdays, the cavities are tightly filled. The formation of contacts of theouter cells of a spheroid with the walls of the substrate andaccordingly the formation of a multilayer tissue-like cell structurecould be observed microscopically as it occurred.

7. Removal of the cells for analysis. Reuse of the substrate

For further analysis the cells anchored in the cavities of a substratecan be dislodged by enzymatic treatment with Trypsin. Alight-microscopic inspection shows that the cells are quantitativelyremoved from the cavities in this manner. A one-week long incubation ofan emptied substrate showed that no cells had remained in the substratecavities. This also demonstrated that the sterility was maintained. Asubstrate from which the cell culture was removed in this manner couldbe reused repeatedly.

What is claimed is:
 1. An apparatus for culturing cells including asubstrate to which said cells selectively attach, said substratecomprising:a) a plate-like body with a lattice-like structure formingopenings separated from one another by side walls, b) a bottom disposedon one side of said lattice-like structure, said bottom being formed bya micro lattice structure having webs with passages formed between thewebs of said micro lattice structure, said webs being about 20 μm thickand the passages having a width of not more than 5 μm, said microlattice structure being permeable for liquids but not for cells, andforming, with said lattice-like structure, cavities for receiving cellsto be grown therein, c) said cavities having a width of between 50 μmand 1000 μm, and d) said bottom consisting of a material which preventsattachment thereto of cells placed into said cavities.
 2. The apparatusaccording to claim 1, wherein the cavities in said lattice structurehave a clear width of between 150 μm and 400 μm.
 3. The apparatusaccording to claim 1, wherein the depth of said cavities for use of thesubstrate with physiological cell cultures is in the range of 50 μm to300 μm.
 4. The apparatus according to claim 1, wherein the depth of saidcavities for use of the substrate with non-physiological cultures isbetween 300 μm and 1000 μm.
 5. The apparatus according to claim 1,wherein, at the side of the lattice structure opposite said bottom, saidside walls have a width in the range of 15 μm to 115 μm.
 6. Theapparatus according to claim 1, wherein the material of which saidsubstrate is made is transparent.
 7. The apparatus according to claim 6,wherein said substrate is mounted into a microscope object carrier.